Int J Clin Exp Med 2020;13(9):7060-7070 www.ijcem.com /ISSN:1940-5901/IJCEM0112790 Original Article The association between the EPHA3 gene polymorphism and non-syndromic cleft lip with or without palate in the western Han Chinese population Yulan Zhou1*, Jiayu Shi2*, Bing Shi1, Zhonglin Jia1 1State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of Cleft Lip and Palate, West China Hospital of Stomatology, Sichuan University, Chengdu, China; 2Division of Growth and Development and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, USA. *Equal contributors. Received April 29, 2020; Accepted July 17, 2020; Epub September 15, 2020; Published September 30, 2020 Abstract: Non-syndromic cleft lip with or without palate (NSCL/P) is a common craniofacial congenital disease which results from multiple susceptibility genes and adverse environmental factors. A recent study reported that the T al- lele of rs7650466 of the EPHA3 gene is a genetic risk factor in the etiology of NSCL/P among the northern Chinese Han population. This study aimed to evaluate the association between the EPHA3 gene variations and NSCL/P in the western Han Chinese population. Here, we conducted targeted region sequencing around rs7632427 (EPHA3) among 159 unrelated NSCL/P cases from the western Han Chinese population and performed a gene-based burden analysis on the rare variations and a single variation association analysis on the common SNVs (single nucleotide variants). Then we found 438 SNVs and 120 indels in all. A burden analysis showed no statistical signifi- cance. The association analysis results suggested that the common SNV rs13094064 was associated with NSCL/P (P=1.86E-07 and OR=0.17), and rs7632427 was most closely related to NSCLO (P=7.49E-05 and OR=0.315). Our study primarily confirmed that the EPHA3 gene was associated with NSCL/P in the western Han Chinese population, making available scientific evidence for future research and genetic counseling. Keywords: Non-syndromic cleft lip with or without palate, EPHA3 gene, single nucleotide variants (SNVs), single nucleotide polymorphism (SNPs) Introduction of multiple genetic mutations and environmen- tal risk factors compared with SCL/P [3, 4]. Cleft lip with or without palate (CL/P) is one of Unfortunately, once a baby is born with NSCL/P the most common oral and craniofacial con- disease, the family faces lots of problems, such genital diseases, with an incidence of one in as feeding difficulties, speech loss, craniofacial every 700 newborns worldwide. In China, the deformity, psychological barriers, and social incidence rate is about 1.67%, including 2.7% integration problems, etc. and also bears for cleft palate only (CPO), 5.6% for cleft lip only severe economic burden because of the need (CLO) and 8.2% for cleft lip and palate (CLP) [1, for multiple surgeries and multidisciplinary inte- 2]. According to its clinical phenotypic charac- grated sequence therapies [5]. Despite the teristics, it can be divided into syndromic cleft rapid development of modern medicine, the lip with or without palate (SCL/P) and non-syn- long and painful treatment process of NSCL/P dromic cleft lip with or without palate (NSCL/P) is always unsatisfactory for the patients. So, it [3]. The approximately 70% of the CL/P patients is urgent to deeply explore the pathogenesis of who have no apparent deformity of other parts this birth defect and strive to provide some sci- of the body are collectively known as NSCL/P. entific theoretical basis for the early diagnosis Moreover, NSCL/P presents a more complicat- and treatment of NSCL/P at the level of molec- ed pathogenesis due to the combined effects ular genetics. EPHA3 is associated with the pathogenesis of NSCL/P Many studies have been carried out on the etio- tion and neural tube formation, etc. [5, 14, 15]. logical mechanism of NSCL/P. Genome-wide GWAS once identified the SNP of rs763242 at association studies (GWAS) have provided va- EPHA3 which was associated with NSCL/P pati- luable guidance for researchers to capture ents (Asian & European) and found that EPHA3 new susceptibility loci and genes of NSCL/P played an important regulatory function in the [6-9], such as MAFB and ABCA4 [6], rs80493- development of cranial and maxillofacial struc- 67 between CREBBP and ADCY9 and loci at ture [8, 9]. Before this, Maunakea et al. [16] 1q32.2, 2q35, 10q25.3, 17p13.1 and 20q12 confirmed that there was a strong methylation [7], rs7590268, rs7632427, rs12543318, and site approximately 200 bp downstream of rs1873147 [8], 1p36, 2p21, 3p11.1, 8q21.3, rs7632427 near EPHA3. This was a valuable 13q31.1, and 15q22 [9]. Obviously, GWAS we- discovery for the functional study of the EPHA3 re widely performed in studying the pathogen- gene and NSCL/P. Recently, Chen et al. [5] dis- esis of complex genetic diseases due to the covered that the SNP rs7650466 of EPHA3 advantages of high efficiency, sensitivity, and gene was associated with NSCL/P in the nor- wide range, but GWAS were also limited by ra- thern Chinese population and also confirmed cial and regional differences. So it is under- that rs7650466 was associated with NSCL/P, stood that the verified P values of the suscep- but not NSCPO, after a stratified analysis. But tible genes associated with NSCL/P varied there is no other study that confirms the asso- among the different countries and ethnic gr- ciation between the EPHA3 gene variations oups, which also indicated that the correlation and NSCL/P among the western Han Chinese strength was different. Based on the previous population. studies, our research team also conducted a In this study, we performed a targeted deep series of relevant studies among the western sequencing based on the haplotype (hg19: Han Chinese population as follows. Jiang et al. chr3:89,412,794-89,552,876) around rs7632- [10] once revealed a strong association bet- 427 (EPHA3) to elucidate the relationship bet- ween the allele G at SNP rs4791774 of the ween the EPHA3 gene and the occurrence of NTN1 gene and NSCLO and further enhanced NSCL/P in the western Han Chinese popula- the evidence in the etiology of NSCL/P. In view tion (Figure 1). of the research based on the MAFB gene, a genotypic transmission-disequilibrium test Materials and methods (TDT) analysis confirmed that the C/C homozy- gote at rs17820943 and T/T homozygote at Samples rs13041247 were over-transmitted and both the C allele at rs17820943 and the T allele In this study, 159 unrelated NSCL/P patients at rs13041247 were over-transmitted among (including 79 cases of NSCLO and 80 cases of NSCL/P patients, suggesting that the MAFB NSCLP) from the western Han Chinese popula- gene was a susceptibility gene for NSCL/P [11]. tion (Table 1), including Chongqing, Sichuan, Other studies also confirmed that rs12543- Guizhou, and Yunnan provinces etc., were col- 318 was associated with NSCLO and that the lected at the Department of Cleft Lip and Pa- allele A at rs7078160 of the VAX1 gene was a late Surgery, West China College of Stomatolo- risk factor for NSCL/P [12, 13]. However, there gy, Sichuan University. All the enrolled patients was no evidence associated with the EPHA3 were diagnosed by a professional physician gene among NSCL/P patients in the western team to rule out deformities in other parts of Han Chinese population. the body. To perform the case-control burden analysis and the association analysis, we used EPHA3, located on the 3p11.1 chromosome, 542 normal individuals’ WGS data from the belongs to the ephrin receptor subfamily of Novogene internal database (http://www.novo- the protein-tyrosine kinase family. Receptor gene.com/) as controls. All of the subjects or tyrosine kinase (RTK) signaling pathways are their guardians read and signed the informed both functionally conserved and diverse, and consent before enrolling in this study, and the regulate cell-cell physiological interactions experimental study program was approved by such as cell migration guidance, axon pathway the Hospital Ethics Committee of West China guidance for angiogenesis, stem cell mainte- Hospital of Stomatology, Sichuan University nance and metastasis, neural network forma- (WCHSIRB-D-2016-012R1). 7061 Int J Clin Exp Med 2020;13(9):7060-7070 EPHA3 is associated with the pathogenesis of NSCL/P Figure 1. The targeted sequencing region of rs7632427 at EPHA3 Hg18. 7062 Int J Clin Exp Med 2020;13(9):7060-7070 EPHA3 is associated with the pathogenesis of NSCL/P Table 1. Type of non-syndromic cleft lip with or without a palate variation type, conservative pre- Case Unilateral Family diction and other information Cleft type Bilateral Male Female Unknown Right Left history were recognized through various databases, such as dbSNP, NSCLO 46 33 0 29 37 13 0 1000 Genome, HGMD, CADD, NSCLP 56 21 3 27 29 24 2 and ExAC. The gene transcript NSCL/P 102 54 3 56 66 37 2 annotation databases, such as Total 204 108 6 112 132 74 4 RefSeq, Consensus CDS, Ense- Note: NSCL/P, non-syndromic cleft lip with or without a cleft palate (NSCLO & mbl and UCSC, were used for NSCLP); NSCLP, non-syndromic cleft lip with a cleft palate; NSCLO, non-syndrom- ic cleft lip only. their exonic variations to deter- mine the amino acid alternation, and the protein functional pre- DNA extraction diction for the nonsynonymous SNVs was per- formed using PolyPhen-2, CADD, SIFT, and Peripheral venous blood was collected from the MutationTaster. patients, and their DNA was extracted using the salting out method. Then it was stored in a Tris- Statistical analysis EDTA buffer in a freezer kept at -80° C.